The aims of this project are to obtain a novel dataset of nociceptin action and ORL receptor selectivity using amphibians in behavioral, binding and molecular studies. The overall goal of is correlate the pharmacological selectivity of nociceptin, to the molecular structure of the nociceptin (ORL) receptor. Recent results using this comparative approach to understanding opioid receptor structure and function in the Northern grass frog, Rana pipiens gave insight on the evolution of opioid receptor types (mu, delta, and kappa), the receptor domains that determine opioid type-selectivity, and on the selection towards greater selectivity in vertebrate opioid receptors. As nociceptin is most closely related to endogenous opioid peptides and the sequence of ORL receptor is most homologous to opioid receptors, data from the proposed studies are needed to complete the comparative analysis of opioid and nociceptin pharmacology and their closely related receptor proteins. The specific aims are to: 1) determine the effects of intraspinally administered nociceptin, nociceptin analogs and selective nociceptin and opioid antagonists on nociceptive thresholds using the acetic acid test, 2) characterize the affinity and density of nociceptin binding sites in spinal cord tissue and the displacement of labeled nociceptin by nociceptin and selective opioid ligands, 3) clone and sequence the ORL-like receptor expressed in the amphibian (rpORL) and characterize the rpORL receptor in transfected cell lines, and 4) perform a comparative analysis of rpORL with respect to existing vertebrate ORL receptors and integrate with similar analyses of vertebrate opioid receptors. As our lab is the sole purveyor of opioid pharmacology using an earlier-evolved vertebrate model, the proposed studies are a unique examination of the functional evolution of opioid and opioid-like receptors. The significance of this project is an increased understanding of opioid and ORL receptor selectivity and the molecular determinants of receptor domains that contribute to pharmacological selectivity. This knowledge will contribute to the detection and the design of more (or less, as the case may be) selective pharmacological agents targeted at opioid and ORL receptors. This will lead to improved therapies for opioid and nociceptin system activation in pathological states and for the treatment of opioid addiction.